Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy

Abstract

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

The expression and mutation of <i>BMPR1B</i> and its association with litter size in small-tail Han sheep (<i>Ovis aries</i>)

“…A large number of studies have shown that the BMPR1B gene plays an active role in mammalian embryo development and follicular development (Tang et al, 2019;Yao et al, 2019; Abdurah- Note that different lowercase letters in the same column represent a significant difference (P < 0.05). man et al, 2019;Zhao et al, 2016). However, further studies are required to deeply investigate the relationship between BMPR1B and mammalian reproduction.…”

A model of mucopolysaccharidosis type IIIB in pigs

“…1 A,B). The founder animal is known to have two insertion sites of the exogenous gene ( BMPR-1B ): one at the NAGLU locus on SSC12 and the other at the CMTM8 locus (within the first intron of the CMTM8 gene) on SSC13 ( Zhao et al, 2016 ). Primers F2/R2 and F3/R3 spanning the vector and host genome ( Fig.…”

“…Genomic DNA was extracted from ear samples using phenol/chloroform method. Primers F1/R1 ( ) were implemented to identify transgenic and non-transgenic (WT) pigs as previously reported ( Zhao et al, 2016 ). Primers F2/R2 and F3/R3 ( ) were used to classify the transgenic individuals with one or two insertion sites.…”

“…In our previous study, we obtained one transgenic founder boar via somatic cell nuclear transfer technology ( Zhao et al, 2016 ). This individual had an insertion of one copy of an exogenous gene ( BMPR-IB ) into exon 6 of the NAGLU gene on pig chromosome (SSC) 12 ( Zhao et al, 2016 ). This insertion disrupted the open reading frame of NAGLU and possibly resulted in a pig model of MPS IIIB.…”

An exonic insertion in the NAGLU gene causing Mucopolysaccharidosis IIIB in Schipperke dogs

“…Mucopolysaccharidosis IIIB (also known as Sanfilippo syndrome B) is caused by variants in the alpha-N-acetylglucosaminidase (NAGLU) gene and has been previously characterized at the clinical and molecular level in humans, emus 11 , cattle 12 , knockout mice 13 , and transgenic swine 14,15 . Furthermore, MPS IIIB has been clinicopathologically reported in Schipperke dogs (OMIA 001342-9615) 10 .…”