BRIEF COMMUNICATION

High-level expression of bioactive recombinant humanlysozyme in the milk of transgenic mice using a modifiedhuman lactoferrin BAC

Shen Liu • Xiangqing Li • Dan Lu • Shengzhe Shang •

Meili Wang • Min Zheng • Ran Zhang • Bo Tang •

Qiuyan Li • Yunping Dai • Ning Li

Received: 31 March 2011 / Accepted: 6 July 2011 / Published online: 30 July 2011

� Springer Science+Business Media B.V. 2011

Abstract Transgenesis has been used for express-

ing human lysozyme (hLZ) in the milk of livestock to

improve their disease resistance. Here we describe a

human lactoferrin (hLF) BAC as a candidate vector

for high-level expression of hLZ in the milk of

transgenic mice. Using recombineering, hLF genomic

DNA in the hLF BAC was replaced by the hLZ gene

(from the ATG start codon to the TAA stop codon),

and flanking regions of the hLF gene (a 90-kb 50 and

a 30-kb 30) were used as transcriptional control

elements for hLZ expression. When this construct

was used to generate transgenic mice, rhLZ was

highly expressed in the milk of four transgenic mouse

lines (1.20–1.76 g/L), was expressed at a lower level

in one additional line (0.21 g/L). rhLZ from the milk

of these transgenic mice exhibited the same antibac-

terial activity as native hLZ. Our results suggest a

potential approach for producing large amounts of

hLZ in the milk of livestock.

Keywords Recombinant human lysozyme (rhLZ) �Recombineering � Human lactoferrin bacterial

artificial chromosome (hLF BAC) � Transgenic mice �Milk

Introduction

Since the production of the first transgenic mice

(Gordon et al. 1980), considerable progress has been

made in applying transgenic technology to livestock

for agriculture, mainly resulting in increases in

disease resistance (Whitelaw and Sang 2005), growth

rate (Hammer et al. 1985; Pursel et al. 1989), carcass

composition (Lai et al. 2006), milk production

(Brophy et al. 2003; Wang et al. 2008), and wool

production (Bawden et al. 1998) as well as reduced

environmental impact (Golovan et al. 2001). Regarding

disease resistance, genetic modifications offer several

possible strategies, including transfer of interfering

RNAs and genes for major histocompatibility com-

plex, T-cell receptors, and specific disease resistance

(Muller and Brem 1991; Whitelaw and Sang 2005). A

prominent example is the demonstration that trans-

genic dairy cattle expressing lysostaphin acquired

resistance to the mastitis-causing bacteria Staphylo-

coccus aureus (Kerr et al. 2001; Wall et al. 2005).

Lysozyme is an antimicrobial molecule that con-

fers disease resistance and shows strong antimicrobial

activity towards gram-positive bacterial species.

Electronic supplementary material The online version ofthis article (doi:10.1007/s11248-011-9536-4) containssupplementary material, which is available to authorized users.

S. Liu � X. Li � D. Lu � S. Shang � R. Zhang �B. Tang � Q. Li � Y. Dai � N. Li (&)

State Key Laboratory for Agrobiotechnology, China

Agricultural University, Beijing 100193, China

e-mail: ninglcau@cau.edu.cn

M. Wang � M. Zheng

GenProtein Biotech Ltd., Beijing 100193, China

123

Transgenic Res (2012) 21:407–414

DOI 10.1007/s11248-011-9536-4

In higher animals, lysozyme exists naturally in avian

egg whites and biological fluids including tears,

saliva, and milk. Lysozyme functions by hydrolyzing

the glycosidic b-(1-4) linkage between N-acetylmu-

ramic acid and N-acetylglucosamine of the peptido-

glycan polymer in the bacterial cell wall (Maidment

et al. 2009). Among the different types of lysozyme,

human lysozyme (hLZ) has a relatively high anti-

bacterial activity, about three times higher than that

of egg white lysozyme. hLZ as a nonspecific immune

factor in human milk plays important roles in

protecting against bacteria, viruses and fungi

(Lee-Huang et al. 1999; Nakajima et al. 1997). When

added to infant formula, hLZ takes part in passive

immunity and reduces microbial infections in the

gastrointestinal tract of breast-fed infants (Lonnerdal

2003; Huang et al. 2002). Human milk has abundant

lysozyme (0.4 g/L), while the milk of other species

contains lower concentrations of lysozyme (e.g., 0.13

and 0.065 mg/L in cows and pigs, respectively)

(Chandan et al. 1964; Maga and Murray 1995). It

has been reported that recombinant hLZ (rhLZ)

produced by transgenic rice significantly improves

the feed efficiency of poultry (Humphrey et al. 2002).

Recently, rhLZ from transgenic dairy goats has been

shown to inhibit the growth of bacteria that cause

mastitis (Maga et al. 2006a). In addition, the

consumption of pasteurized goat milk containing

rhLZ has the potential to improve gastrointestinal

health and is protective against an enteropathogenic

Escherichia coli (EPEC) infection in young weaned

pigs (Brundige et al. 2008). Thus, the expression of

rhLZ in milk may enhance resistance to mastitis in

cows and protect young pigs as pigs used in Brundige

et al. (2008) were weaned from diarrheal disease.

Our previous work has shown that the plasmid

pBC-hLZ using goat b-casein gene promoter can be

used to express bioactive rhLZ in the milk of

transgenic mice, and the highest level was up to

1.4 g/L, albeit at very different levels among indi-

viduals (Yu et al. 2006). This construct was also

transfected into primary female porcine fetal fibro-

blasts, and transgenic cloned pigs were generated, but

the expression of rhLZ was very low (0.32 mg/L) (Li

et al. 2009; Tong et al. 2010). Similar results were

obtained with transgenic cattle expressing rhLZ

(25.96 mg/L) (Yang et al. 2011). Thus, optimization

of the hLZ expression construct is required. We also

screened a series of transgenic mice expressing a

BAC clone for expression of human lactoferrin

(hLF), and this BAC clone allowed consistent

expression of high levels of recombinant human

lactoferrin (rhLF) in the milk of transgenic mice

(highest expression reached 8 g/L) (Liu et al. 2004).

Four years later, somatic cell nuclear transfer (SCNT)

was used to produce transgenic cloned cows that

secrete rhLF at high levels (2.5 and 3.4 g/L) (Yang

et al. 2008). Thus, we speculate that the hLF BAC

construct is a candidate expression vector that will

allow high levels of hLZ expression.

In this study, the hLF locus of the hLF BAC

construct was replaced with the 4.8-kb hLZ gene by

recombineering. The 90-kb 50 and 30-kb 30 flanking

regions of the hLF gene were used as the transcrip-

tional control elements for hLZ expression. A neo-

mycin resistance gene that works in both prokaryotic

and eukaryotic expression systems was inserted in

front of the 50 flanking region of the hLF BAC as the

selectable marker for SCNT. Transgenic mice were

generated with this construct, and rhLZ was

expressed at high levels (1.20–1.76 g/L) in the milk

of four transgenic lines. Only one line expressed

rhLZ at low levels (0.21 g/L). The rhLZ from

transgenic milk exhibited the same antibacterial

activity as native hLZ. Our results suggest a potential

approach for producing large amounts of hLZ in the

milk of livestock that could be purified as a

nutraceutical.

Results

Construction of pBAC-hLF-hLZ-Neo

expression vector

Figure 1 shows the strategy for replacing 28.9 kb of

hLF genomic sequence with 4.8 kb of hLZ genomic

DNA on the hLF BAC. The procedure of modifying

the pBAC-hLF-hLZ-Neo construct was divided into

three distinct steps, which were each verified by PCR

and sequencing with the primers listed in Table S1.

The first step is a replacement of hLF coding

sequence with the one for hLZ, thus the targeting

construct pMD19-hLZ-Zeo was linearized and elec-

troporated into competent SW102 cells containing

hLF BAC for recombination. Ten zeocin resistant

clones were picked and verified by PCR with hybrid

primers P1 (Table S1), which bind to the hLF 50

408 Transgenic Res (2012) 21:407–414

123

flanking regions and hLZ genomic sequence, respec-

tively. All ten clones produced a band of the expected

length (703 bp). No product was amplified from

unmodified hLF BAC (Fig. S1a). The PCR-positive

SW102 bacterial cells were made competent for the

next procedure. In the second step, the neomycin

resistance cassette, which allows stably transfected

eukaryotic cells to be selected using G418 and

expresses kanamycin resistance in E. coli, was

inserted into the hLF BAC as a selectable marker

for future SCNT. Recombineering was performed and

verified as described above. The amplified products

were 2,541 bp for BAC clones that recombination

occurred at the correct location, and 1,960 bp for the

negative control (Fig. S1b). In the final step, the

zeocin cassette flanked by two direct repeat FRT sites

was removed after transient expression of FLP

recombinase in E. coli strain SW102. PCR analysis

was performed across the recombination site, and the

amplified products of positive clones were found to

be 1,129 bp, about 1 kb smaller than the negative

control because of the deletion of a zeocin cassette

(Fig. S1c). P4 primers were prepared for digoxigenin

(DIG)-labeled probes for Southern blot analysis

(Table S1). The final modified pBAC-hLF-hLZ-Neo

construct contained a 90-kb 50 flanking region of the

hLF gene, the 4.8-kb hLZ genomic fragment with a

single FRT site retained, a 30-kb 30 flanking region of

the hLF gene, and a Neo cassette for future SCNT.

Generation of transgenic mice using modified

BAC clone

Circular pBAC-hLF-hLZ-Neo BAC DNA was micro-

injected into pronuclei of fertilized eggs, and trans-

genic mice were generated by standard microinjection

(Hogan et al. 1994). Two female (no. 32, 39) and three

male (no. 26, 30, 35) transgenic founders were

obtained from 41 mice analyzed by PCR (Fig. 2a;

efficiency = 12.2%). Southern blotting was further

used to confirm that the transgenes were integrated

into the genome of transgenic mice. A 637-bp DIG-

labeled DNA probe was produced by PCR using

pBAC-hLF-hLZ-Neo construct DNA as a template

Fig. 1 Diagrammatic representation of BAC modification.

The recombineering procedure was divided into three distinct

steps. In step 1, the 4.8-kb hLZ genomic sequence (from the

ATG start codon to the TAA stop codon) flanked by two

homology arms was obtained by PCR, and a Zeo cassette

flanked by two FRT sites for positive selection was ligated into

intron 2 of the hLZ gene. The linear targeting substrate DNA of

the hLZ-Zeo gene was electroporated into competent SW102

cells containing hLF BAC. In step 2, a Neo cassette was

flanked by two loxP sites and designed to be inserted into the 50

genomic DNA of hLF BAC for drug selection of cultured

somatic cells. In step 3, the Zeo cassette was excised by

transformation of a helper plasmid encoding the FLP recom-

binase, and a single FRT site was retained after recombination.

The positions of primers (P1, P2, P3, and P4) used for PCR

verification or Southern blot probes are indicated by arrows

Transgenic Res (2012) 21:407–414 409

123

and a PCR reaction mixture containing DIG-dUTP.

The result of Southern analysis was consistent with

that of PCR (Fig. 2b). Upon sexual maturity, the

transgenic founders were mated with wild-type mice,

and all founders transmitted the transgenes to their

offspring.

Expression of rhLZ

Transgene expression in transgenic female mice was

assessed by RT-PCR using RNA isolated from

lactating mammary glands (day 13) and seven other

tissues (heart, liver, spleen, lung, kidney, stomach,

and intestine) of two lines (F1 generation of line 26

and 39). The mouse GAPDH gene was used as a

control for mRNA extraction and loading. The

primers Exon1-2-F and Exon4-R (Table S1) were

designed based on the hLZ coding sequences and

yielded a 322-bp PCR product. The forward primer

was designed to cross one intron. Strong amplification

was obtained with mRNA from lactating mammary

glands, whereas very low levels of transcripts were

detected in heart, liver, spleen, lung, kidney, and

intestine, and no transcript was detected in stomach

(Fig. 2c). Both line 26 and 39 had shown the same

pattern of mRNA expression.

High expression of rhLZ in milk

of transgenic mice

To characterize rhLZ expression, F0 females and F1

females from F0 male were used, and milk samples

from all transgenic females were collected during

mid-lactation (days 9–16). Samples were diluted and

analyzed by western blot analysis. Milk from wild-

type mice was used as a negative control, and 0.5 lg

natural hLZ standard was used as a positive control.

Most transgenic lines showed a strong signal at

14.7 kDa, indicating high-level expression of rhLZ in

milk; no band was observed in the negative control

Fig. 2 Molecular characterization of transgenic mice. a PCR

detection of transgenic founders. M 100-bp DNA ladder, PCpositive control using pBAC-hLF-hLZ-Neo construct, NCnegative control; transgenic founders numbered 26, 30, 32,

35, and 39. The amplified products are 703 bp for the hLF-

hLZ hybrid gene. b Southern blot analysis of transgenic

founders. EcoRI-digested genomic DNA was hybridized with

digoxigenin-labeled probe of amplified hLZ fragments. PCpositive control, NC genomic DNA of non-transgenic mice as a

negative control. c RT-PCR analysis transgene expression in

transgenic mouse tissues. The 322-bp product was abundant in

lactating mammary gland of transgenic mice, and weak signal

was detected in six out of seven other tissues. M 100-bp DNA

ladder; M mammary gland tissue of transgenic line 26 on day

13 of lactation; H heart, Li liver, Sp spleen, Lu lung, K, kidney,

St stomach, I intestine, NC mammary gland tissue of non-

transgenic mouse. The mouse GAPDH gene was used as a

positive control for mRNA extraction and loading

410 Transgenic Res (2012) 21:407–414

123

(Fig. 3). The concentration of rhLZ was further

quantified by ELISA, which showed that four of the

five transgenic lines had high-level, consistent

expression of rhLZ ranging from 1.2 to 1.74 g/L.

By both western blotting and ELISA, only no. 32 had

a low level of expression (0.2 g/L; Table 1).

Assessment of rhLZ antibacterial activity

Two methods were used to analyze the bactericidal

activity of rhLZ in M. lysodeikticus, because this

organism is very sensitive to lysozyme. The lysoplate

method is a convenient method for analyzing bacte-

ricidal activity and for roughly estimating the

expression level of rhLZ in the milk. Milk samples

were spotted on individual circles of quantitative

filter paper, which were placed on an agar plate

containing M. lysodeikticus. After incubation at 28�C,

transparent zones around the filter paper circles

indicate lysis of the bacteria. Transparent zones

around filters containing natural hLZ standard or milk

from transgenic mice were clearly visible after

incubation for 36 h (Fig. 4). No transparent zone

was formed with milk from a non-transgenic mouse.

The activity of rhLZ can be roughly compared by

measuring the diameter of the clear zone. Consistent

with the western blotting and ELISA results, a

smaller zone of transparency was seen for line 32.

Milk samples were quantitatively examined using

a turbidometric assay. In this assay, the enzymatic

activity of hLZ is determined by monitoring the

reduction in turbidity of a suspension of M. lys-

odeikticus cells at 450 nm. A standard curve was

established using hen egg-white lysozyme (HEWL)

ranging from 1,000 to 8,000 U/lL, and then the rhLZ

activity of milk samples were quantified. The

antibacterial activity in milk from four transgenic

lines ranged from 3,137 to 3,498 U/lL; the level for

line 32 was considerably lower (872 U/lL; Table 1).

Milk from non-transgenic mice and the natural hLZ

standard showed activity of 155 ± 194 U/lL and

1,528 ± 275 U/lL, respectively.

Discussion

The hLF-hLZ hybrid BAC was constructed to induce

high expression of rhLZ in mammary glands. This

vector was designed for high-level, consistent expres-

sion in the milk of transgenic mice and yielded the

highest level of rhLZ expression reported to date.

Considerable effort to optimize levels of protein

expression has concentrated on overcoming chromo-

somal position effects and increasing the length of the

Fig. 3 Western blot detection of recombinant hLZ expressed

in milk of transgenic mice. Milk samples were diluted 1:10 in

PBS, and 3 lL of each sample was separated by SDS–PAGE

under reducing conditions. PC, 0.5 lg natural hLZ standard

(Sigma; 14.7 kDa); NC, milk of non-transgenic mice as a

negative control; 32 and 39 are milk from female founder mice;

30-11, 30-13, 35-17, and 26-22 are milk from different female

F1 offspring of male founders

Table 1 Expression of rhLZ in milk of transgenic mouse lines

Line Sex of founder rhLZ expression

levela in F0 (g/L)

rhLZ expression

levela in F1 (g/L)

Antibacterial

activityb (U/lL)

Germ line

transmission

26 Male 1.74 ± 0.15 3,498 ± 285 Yes (5/10)

30c Male 1.36 ± 0.01

1.33 ± 0.07

3,228 ± 253

3,137 ± 125

Yes (2/10)

32 Female 0.21 ± 0.01 872 ± 145 Yes (1/9)

35 Male 1.25 ± 0.06 3,397 ± 253 Yes (7/13)

39 Female 1.20 ± 0.18 3,329 ± 222 Yes (1/8)

a The concentration of rhLZ was quantified by ELISA in female founders or in F1 female offspring from male foundersb Antibacterial activity was determined by turbidometric assayc The line 30 founder mouse fathered two female offspring (F1: 30-11 and 30-13)

Transgenic Res (2012) 21:407–414 411

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regulatory elements. An hLF bacterial artificial

chromosome was chosen as a candidate expression

vector. Artificial chromosomes generally contain all

the regulatory elements necessary for exogenous gene

expression as well as specific sequences such as

scaffold/matrix-attachment regions and locus control

regions or insulators that are likely to overcome

positional effects and thus increase transgene expres-

sion (Giraldo and Montoliu 2001). Three types of

artificial chromosomes are commonly used in trans-

genic studies: yeast artificial chromosomes (YACs),

BACs, and P1-derived artificial chromosomes

(PACs). BACs are considered the best type of

expression vector because they can adapted to most

protocols, can accommodate genomic inserts up to

300 kb, have a low frequency (\5%) of chimerism,

and are more stable (Giraldo and Montoliu 2001).

Transgenic mice created using a goat lactalbumin

BAC showed position-independent and copy number-

related expression (Stinnakre et al. 1999). Our

previous results also demonstrated that the hLF

BAC provided high-level, consistent expression of

rhLF in transgenic mice and cattle (Liu et al. 2004;

Yang et al. 2008). Thus, we chose hLF BAC as the

candidate vector for high expression of rhLZ in the

mammary gland.

Mammary gland transgenesis of hLZ was first

reported by Maga et al. (1994). rhLZ was expressed

in the milk using either 20 kb of bovine as1-casein

promoter, resulting in concentrations that varied from

0.25 to 0 0.71 g/L (Maga et al. 1995), or 3.7 kb of

bovine b-casein promoter, resulting in no detectable

mRNA expression in mammary tissue. A possible

reason for this was that the latter construct lacked

some essential regulatory elements. Usually, 20 kb of

promoter is sufficiently long to contain all regulatory

elements required for high-level expression. How-

ever, a possible disadvantage of the former construct

is that a cDNA was used rather than genomic DNA,

which can lower hLZ expression because introns

increase transcriptional efficiency (Brinster et al.

1988). In 2006, hLZ-expressing transgenic goats

were generated by standard pronuclear microinjec-

tion, and the expression level of rhLZ in the milk was

0.27 g/L, or 68% of the level found in human milk

(Maga et al. 2006b). In our laboratory, transgenic

hLZ mice produced milk at levels up to 1.4 g/L (Yu

et al. 2006), but the level of expression was not

similar among different lines. hLZ-expressing trans-

genic pigs or cows generated by SCNT also had very

low expression levels (Tong et al. 2010). One reason

for the transgene expression variability may be

attributed to a chromosomal position effect (Clark

et al. 1994), although two chicken b-globin insulators

were used. A second reason may be that the goat

b-casein promoter in the pBC1 vector was only

Fig. 4 Lysoplate assay for lytic activity of rhLZ against

M. lysodeikticus. Small white circles are 6-mm quantitative

filter papers spotted with 10 lL of 1:10 diluted milk sample.

Transparent zones around the quantitative filter papers indicate

bacterial lysis. PC0.5, 0.5 lg natural hLZ standard; PC1.0,

1 lg natural hLZ standard; NC, milk of non-transgenic mice as

a negative control; 32 and 39 are milk from female founder

mice; 30-11, 30-13, 35-17, and 26-22 are milk from different

female F1 offspring of male founders

412 Transgenic Res (2012) 21:407–414

123

4.1 kb, which works well in transgenic mice but may

be not long enough to contain all the mammary

regulatory regions in pigs or cows.

In this study, hLF genomic DNA in the hLF BAC

was replaced by the full-length hLZ gene by

recombineering, and the resulting circular BAC was

used to generate transgenic mice. The overall

efficiency of transgenesis was 12.2%, which is

comparable to that of standard DNA constructs

(5–20%) and of linearized BACs (Liu et al. 2004).

Our results were consistent with the findings of others

(Van Keuren et al. 2009). The transmission of the

transgenes was detected by analyzing tail DNA. Of

the 50 F1 pups tested, 16 were positive for transgene

(Table 1).

The amount of rhLZ in the milk was quantified by

ELISA, which showed that four out of five transgenic

mouse lines had a high level of expression, demon-

strating that this BAC vector provided very consistent

expression. Only line 32 showed low expression. No

direct relationship was observed between the trans-

gene copy number and the amount of rhLZ (Supple-

mentary material 2), which is inconsistent with a

previous finding (Stinnakre et al. 1999). Our results

indicate that the pBAC-hLF-hLZ-Neo construct con-

tained long-range regulatory elements and partially

overcame any chromosomal positional effects when

integrated into the mouse genome. Furthermore, the

use of genomic DNA likely enhanced the expression

level of rhLZ compared to cDNA.

Transgene expression in transgenic female mice

was assessed by RT-PCR in mammary gland and

seven other tissues. hLZ transcripts were abundant in

mammary gland tissue of lactating mice, but weak

signals were also detected in most other tissues

tested. This ectopic expression was not unexpected

because the hLZ gene was under the control of the

hLF promoter, the activation of which is constitutive

but is differentially regulated through multiple sig-

naling pathways (Teng 2002). More experiments will

be needed to examine whether the ectopic expression

of hLZ affects the health of transgenic animals.

A well-known property of lysozyme is its lytic

activity against gram-positive bacteria, especially

M. lysodeikticus cells. Therefore, the lytic activity

of rhLZ was assayed in M. lysodeikticus cells by both

turbidometric and lysoplate assays, and rhLZ dis-

played a level of lytic activity that was about two

times higher than that of 1 lg natural hLZ standard.

This indicates that the rhLZ generated by this

expression vector is a good candidate for an antimi-

crobial agent in milk of transgenic animals.

In conclusion, we have successfully generated

transgenic mice expressing high levels of rhLZ in

milk. Our results suggest a potential approach for

producing large amounts of hLZ in the milk of

livestock.

Acknowledgments We would like to thank Drs. Yaofeng

Zhao, Sen Wu, Jia Tong, Guangbin Zhou, Jianwu Wang, Tian

Yu, Jin He and Jianxiang Xu for revising the manuscript,

Hongxing Chen for his discussions on BAC modification, and

Min Zhang, Rui Fang, Mingjun Bi, Zubin Cao, Zhisheng Chen

and Junna He for excellent technical assistance. This work was

supported by ‘‘863’’ High-Tech Research Development

(Project Grant No. 2009AA10Z110 and 2010AA10A103).

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